The migration of cancer cells away from the primary tumor mass and their subsequent metastasis to distant organs is regarded as a fatal step in cancer progression and is associated with the majority of cancer mortalities. Furthermore, individual cancer cells appear to be able to evade current pharmacologic intervention of invasion and metastasis by switching between mesenchymal and amoeboid modes of motility. The cellular mechanisms controlling this phenotypic plasticity are poorly understood. We have recently identified distinct functions for the closely related adhesion-associated scaffold proteins paxillin and Hic-5 in the regulation of tumor cell plasticity, invasion and metastasis. In this proposal, using established cancer cell lines, as well as cells isolated from primary tumors, we will apply state-of-the art real-time imaging techniques to track tumor cell morphology and migration as well as adhesion and cytoskeletal dynamics in 3D-extracellular matrix in vitro model systems. Xenograft studies in mice will be used to evaluate the relative impact of paxillin and Hic-5 signaling on tumor progression and metastasis in vivo. We will use RNA interference and mutant protein expression to dissect the respective roles for paxillin and Hic-5 in controlling the mode of tumor cell invasion and identify the pertinent functional domains and signaling pathways. We will use similar approaches to study a role for paxillin in the regulation of matrix metalloproteinase-2 (MMP-2) trafficking and secretion to control mesenchymal tumor invasion strategies. Hic-5 is upregulated during TGF--induced epithelial mesenchymal transition. The role of Hic-5 in TGF--dependent cell invasion, through the formation and function of matrix-degrading invadopodia, will also be examined. The proposed studies will provide insight into the underlying cellular mechanisms controlling tumor cell migration and invasion and the coordination of their phenotypic plasticity and may in the future suggest novel strategies for detection or treatment of metastatic cancers.